Oligonucleotides belong to a class of biopharmaceuticals with a great potential for therapies of various diseases including cancer, viral infections and inflammatory disease to name a few. An important approach to advancing oligonucleotides as therapeutics involve modifications of the oligomer backbone to provide, among other things, metabolic resistance, chemical stability and to improve in vivo transport to the site of action. Examples of modified backbone chemistries include: peptide nucleic acids (PNAs) (see Nielsen, Methods Mol. Biol., 208:3-26, 2002), locked nucleic acids (LNAs) (see Petersen & Wengel, Trends Biotechnol., 21(2):74-81, 2003), phosphorothioates (see Eckstein, Antisense Nucleic Acid Drug Dev., 10(2):117-21, 2000), methylphosphonates (see Thiviyanathan et al., Biochemistry, 41(3):827-38, 2002), phosphoramidates (see Gryaznov, Biochem. Biophys. Acta, 1489(1):131-40, 1999; Pruzan et al., Nucleic Acids Res., 30(2):559-68, 2002), thiophosphoramidates (see Gryaznov et al., Nucleosides Nucleotides Nucleic Acids, 20(4-7):401-10, 2001; Herbert et al., Oncogene, 21(4):638-42, 2002).
In contrast to large selection of reagents available for introducing the unsubstituted sulfur atom to phosphorus esters, the spectrum of groups allowing for sulfurization of H-phosphonate esters with the protected sulfur is limited (e.g. Dreef, et al. Synlett, 481-483, 1990, U.S. Pat. No. 6,506,894). Practically, only the cyanoethylsulfide group has been used extensively in this reaction during the solution synthesis of oligonucleotides with chromatographic purification at each step. Patent Application PCT/EP2009/067902 in the name of the applicant the contents of which is incorporated by reference into the present patent application discloses novel sulfurizing reagents, a process for their manufacture and their use in the economical and convenient synthesis and purification of phosphorothioate oligonucleotides in solution via the H-phosphonate method. A critical problem in the solution synthesis of oligonucleotides concerns the necessity to obtain high substrate conversions with excellent specificity at each synthetic step giving high purity products in a form that facilitates simple purification, in particular avoiding chromatography. Given the lack of methods allowing for economical solution phase synthesis, the solution phase technology does not seem to be currently used for commercial scale oligonucleotide synthesis.
The invention makes now available a new method for the synthesis of oligonucleotides by using novel sulfurizing reagents, which allows for an economical and efficient synthesis and purification of oligonucleotides notably in solution.